The heart is a major locus for the toxic and lethal effects of cocaine. An important action of cocaine in the heart is to inhibit presynaptic catecholamine reuptake, leading to an elevated sympathomimetic state. While this undoubtedly plays a role in the harmful effects of cocaine, it is probable that other factors also contribute to cocaine cardiotoxicity since most sympathomimetic interventions do not have the deleterious effects associated with cocaine use. In preliminary experiments using isolated ventricular heart muscle cells, we have observed a direct action of cocaine to interfere with the cytosolic Ca2+ transients responsible for excitation-contraction coupling. It is proposed that the direct actions of cocaine on cardiomyocytes may contribute to the cardiotoxic effects of cocaine. In particular, the combination of elevated catecholamine levels (which activate specific Ca2+ flux components of the Ca2+ transient), together with the inhibitory actions of cocaine, could lead to abnormalities of excitationcontraction coupling. The objective of the present proposal is to characterize the direct effects of cocaine on the Ca2+ transient and contractility of isolated cardiac myocytes and to elucidate the mechanisms responsible for these effects. In addition, the interactions between cocaine and catecholamines at the level of excitation-contraction coupling will be investigated in the-isolated cardiomyocyte system. The primary experimental approaches will involve the use of fluorescent Ca2+ indicators and a high-speed digital imaging system to follow Ca2+ fluxes in electrically stimulated myocytes at the single cell level. This technique allows us to measure the kinetic parameters and subcellular distribution of these Ca2+ fluxes with millisecond time resolution, while simultaneously monitoring cell contraction. We will also use the whole-cell configuration of patch-clamp to study the effects of cocaine on individual sarcolemmal ion conductances under voltage-clamp. The voltage-clamp and Ca2+ imaging methods will be combined to investigate how the effects of cocaine on ion channels might play a role in the inhibitory effect of cocaine on the Ca2+ transient. The long term objective of the proposed study is to understand the basic mechanisms which underlie the cardiotoxic actions of cocaine and to determine how each of these mechanisms contributes to the harmful effects of cocaine in vivo.